Blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) is widely used for non-invasively mapping brain function in research and clinical settings. In contrast, quantitative BOLD fMRI approaches have not completely fulfilled their research and clinical potentials because important and unverified concerns remain regarding the appropriateness and accuracy of the calibration procedure. The objective in this particular application is to examine unverified central assumptions used in the BOLD calibration procedures: 1) CO2 and O2 challenges used for calibration do not alter a cerebral metabolic rate of oxygen (CMRO2) and 2) the changes in cerebral blood volume (CBV), especially venous CBV, are directly related to the changes in cerebral blood flow (CBF) induced by CO2 and O2 challenges. To attain this objective we will use well- established techniques to directly determine the following parameters: 1) ongoing neural activity, vascular oxygen concentration and CBF changes induced by CO2 and O2 challenges for CMR O2 estimates, and 2) changes in CBF and venous CBV induced by CO2 and O2 challenges as well as by neural stimulation to examine the relationship between CBV and CBF. The rationale for the proposed research is that the quantitative assessment of BOLD fMRI requires more comprehensive investigations of CO2 or O2-induced changes in vascular and metabolic neurophysiology. Once the CO2 and O2 challenges are validated as iso- metabolic stimuli and the relationship between venous CBV and CBF is established, these calibration strategies will be readily applicable for the accurate assessment of human brain function in the research and clinical settings. This approach is innovative because it provides a direct and more comprehensive assessment of unverified assumptions for BOLD calibration. The proposed research is significant because it could ultimately lead to the advancement of quantitative fMRI, including CMRO2 estimates, in the diagnosis and treatment planning of diseases that are associated with tumor, stroke and neurological disorders. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the ultimate establishment of quantitative functional MRI is expected to advance the diagnosis and treatment planning of diseases that are associated with stroke and neurological disorders.